Methods for augmenting immunological responses through the administration of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone-sulfate (DHEA-S)

ABSTRACT

Methods for augmenting immune responses in immunodeficient individuals are disclosed. The methods utilize steroid hormones, particularly DHEA, its prohormones (particularly DHEA-S), and DHEA-cogeners. Additional embodiments of the invention include pharmaceutical compositions for use in the methods.

This invention was made with government support under grant numbers CA22126 and CA 25917 awarded by the Department of Health and HumanServices/Institutes of Health. The government has certain rights in theinvention.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of application Ser. No. 08/219,418,filed Mar. 29, 1994, now abandoned, which is a continuation ofapplication Ser. No. 07/779,499, filed Oct. 18, 1991, now abandoned,which is a continuation-in-part of Application U.S. Ser. No. 07/412,270filed Sep. 25, 1989, now abandoned, the specification of which isincorporated by reference herein in its entirety.

TECHNICAL FIELD

The invention relates to methods and compositions used for theaugmentation of an immune response in vivo and in vitro by the use ofsteroid hormones, more specifically, by the use ofdehydropepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEA-S),and analogs thereof.

BACKGROUND

Immunosuppression in animals can result from a depressed capacity toproduce species of lymphokines which are essential to the development ofprotective forms of immunity. Imbalances between various types oflymphokines, where species of lymphokines capable of promoting one formof immune response exhibit enhanced production, while those lymphokinesneeded to promote protective forms of immunity are suppressed, can alsolead to immunosuppression. Individuals may be immunosuppressed as aconsequence of endogenous elevations in adrenal glucocorticosteroid(GCS) levels. This condition could result from viral infections, fromcertain bacterial infections, certain parasitic infections, cancer, someautoimmune syndromes, and stress and trauma, or as a secondaryconsequence to any clinical condition that causes an elevated productionof interleukin-1 (IL-1). Plasma glucocorticoid steroid levels also canbe elevated exogenously as a consequence of therapeutic treatment for avariety of clinical conditions. In addition to the above, it is alsowell known that certain essential functions of the immune system declinewith age, a situation which correlates with elevations in adrenal outputof glucocorticoid steroids and depressions in production of other typesof adrenal steroid hormones.

It is known that lymphocytes exported from the thymus undergo a seriesof differentiation events which confer upon them the capacity torecognize and respond to specific peptide antigens presentedappropriately in the context of self major histocompatibility complex(MHC) molecules. Mechanistically, thymic maturation is a complex processwhich includes an irreversible rearrangement of T cell receptor genes,the cell surface expression of these gene products as disulfide-linkedheterodimers, positive and negative selection processes to provideappropriate restriction and avoidance of self-reactivity, and thesynthesis and expression of CD4 or CD8 as accessory adhesion molecules.Microenvironmental influences within the thymus play an essential rolein the fidelity of this process.

Subsequent to leaving the thymic microenvironment, mature T lymphocytesgain access to the recirculating T cell pool where they move freely viathe blood between mucosal and nonmucosal lymphoid compartments in themammalian host (Hamann et al. (1989), Immunol. Rev. 108:19).T-lymphocyte expression of lymphoid tissue-specific homing receptors,which are complementary for vascular addressins on high endothelialvenules present in Peyer's patches and peripheral lymph nodes, provide abiochemical means for selectivity to this recirculation process (id.).Non-activated lymphocytes can move freely between mucosal and nonmucosallymphoid tissues due to the presence of both types of homing receptorson their plasma membranes (Pals et al. (1989), Immunol. Rev. 108:111).Effector lymphocytes, and antigen-activated immunoblasts which arestimulated in a particular site in the body, however, exhibit a far moreselective migratory behavior. These cells move primarily to tissuesoriginally involved in antigen exposure and cellular activation (Hamannet al. (1989), supra; Pals et al. (1989), supra.).

An immune response is initiated following T cell recognition of antigenpeptides in the context of self MHC molecules and generally takes placein one of the host's secondary lymphoid compartments. Cellularactivation is triggered by the binding of antigen to the T cell receptor(TCR), forming an antigen/TCR complex which transduces theantigen-specific extracellular stimulation across the plasma membrane,and generates intracellular signals which include the activation ofprotein kinase C and the increases in intracellular calcium. Whilesignal transduction can lead to T cell unresponsiveness, positive signaltransduction events trigger a series of additional biochemicalprocesses. One consequence of this activation is the stimulatedproduction of a number of biologically active molecules which arecollectively termed lymphokines. (See, Alcover et al. (1987), Immunol.Rev. 95:5; Gelfand et al (1987), Immunol. Rev. 95:59).

The lymphokines, many of which function primarily through autocrine andparacrine mechanisms, serve to mediate numerous effector functionscontrolled by T cells through their capacity to regulate cellularproliferation, differentiation, and maturation events in lymphocytes,plus other hematopoietic and somatic tissue cells (Paul (1989), Cell57:521).

Each of the various types of lymphokines exhibit pleiotropic activities,dependent upon the specific type of cellular targets being stimulated.The biological evaluation of recombinant forms of specific lymphokineshas determined that individual species can possess both distinct andoverlapping cellular activities (Paul (1989), supra). Interleukin-2(IL-2) and interleukin-4 (IL-4), for example, share the capacity tofacilitate T cell growth but are disparate in their relativecontribution to cellular and humoral immune responses. Cloned T celllines, restricted in their capacity to produce individual species oflymphokines, have been described which demonstrate unique capabilitiesin serving as effector cells or helper cells for various immuneresponses (Paul (1989), supra; Hayakawa et al. (1988), J. Exp. Med.168:1825; Mossman et al. (1989), Ann. Rev. Immunol. 7:145).

Treatment of individuals for immunosuppression has been focused on theuse of purified lymphokines, usually IL-2, to restore normal propagationof T cells. Illustrative of this are the disclosures of U.S. Pat. No.4,661,447 (issued Apr. 28, 1987 to Fabricus et al.), U.S. Pat. No.4,780,313 (issued Oct. 25, 1988 to Koichiro et al.), and U.S. Pat. No.4,789,658 (issued Dec. 6, 1988 to Yoshimoto et al.). However, thesystemic administration of IL-2 for therapeutic purposes has numerousside effects. These side effects include fever, hypotension, hepatic andrenal failure, myocardial infarctions, capillary leak syndrome, andmassive edema (Dinatello et al. (1987), New England J. Med. 317:940.

Applicants' invention embodies methods for treating immunosuppressionwhich are without the side-effects found with the purified lymphokines.These methods utilize the androgen steroid hormones, more specificallydehydroepiandrosterone (DHEA), the sulfated derivative thereof (DHEA-S),and analogs thereof.

DHEA is steroid hormone that has been extensively studied for manyyears. It has been reported to be involved in a wide variety ofphysiologic, immunologic, and pathologic conditions (for reviews, seeRegelson et al, (1988), Ann. N.Y. Acad. Sci. 521:260; Gordon et al.(1986), Adv. Enzyme Reg. 26:355-382). Most endocrinologists believe thatthe primary function of DHEA is to serve as a precursor for thesynthesis of testosterone and the estrogens by the gonads. Thebiosynthetic relationship of DHEA to other steroid hormones is shown inFIG. 1 (taken from Cook and Beastall in Steroid Hormones, A PracticalApproach (Green and Leake, eds., IRL Press Limited, 1987). Prior to itsrelease into the bloodstream, the vast majority of newly synthesizedDHEA becomes sulfated. The conjugated steroid DHEA-S (shown in FIG. 2),is a secretory product of the adrenal gland in man and certain primates.DHEA-S represents the major steroid hormone in the circulation ofhumans, and is converted to DHEA via a sulfatase.

Therapeutic uses for DHEA and certain analogs have been reported fordiabetes, dry skin, ocular hypertension, obesity, and retroviralinfections. Illustrative of these reports are the disclosures of U.S.Pat. No. 4,395,408 (issued Jul. 26, 1983 to Torelli et al.), U.S. Pat.No. 4,518,595 (issued May 21, 1985 to Coleman et al.), U.S. Pat. No.4,542,129 (issued Sep. 17, 1985 to Orentreich), U.S. Pat. No. 4,617,299(issued Oct. 14, 1986 to Knepper), U.S. Pat. No. 4,628,052 (issued Dec.9, 1986 to Peat), U.S. Pat. No. 4,666,898 (issued May 19, 1987 toColeman et al.), European Patent Application No. 0 133 995 A2 dated Feb.8, 1984 (inventor, Schwartz et al.), and UK Patent Application No. GB 2204 237 A dated Apr. 14, 1988 (inventor, Prendergast).

SUMMARY OF THE INVENTION

It is an objective of the invention to provide a method for enhancingthe biosynthesis of selected lymphokines by activated T cells. Anotherobjective of the invention is to enhance immune functions in warmblooded animals by restoring their capacity to naturally producephysiological concentrations of these lymphokines with a minimization ofside effects. Further objectives of the invention are to provideapplications of the method for clinically diagnosing deficiencies ofinterleukin production, maintaining in vitro tissue cultures of T cells,and overcoming certain types of immunosuppression associated withelevated GCS levels, caused by endogenous production or exogenousadministration. Final objectives of the invention are to provideapplications of the method as a vaccine adjuvant to selectively directthe vaccine-induced immune response down a protective, rather than apotentially pathologic or non-protective, immunologic pathway, as atreatment for naturally occurring ageing-related decreases in immunefunction, as a treatment for stress or trauma-induced decreases inimmune function, and as a means to facilitate desensitization to agentsto which a warm-blooded animal is allergic.

DHEA-S is a prohormone which is naturally converted to DHEA in theperipheral lymph nodes of animals with normal immune function. The DHEAproduced then influences the T lymphocytes within the lymph node andexerts controlling influences on their ability to respond whenactivated. This provides a means to regulate the potential of T cells byfluctuating the degree to which a particular steroid hormone existswithin a particular tissue. Old individuals and/or stressed individuals,including humans, lose the capacity to produce DHEA-S, resulting inaltered T-cell responsiveness. Various embodiments of the inventionrestore the metabolite produced from DHEA-S in the anatomic compartmentin which T-cell responsiveness is required for normal immune responsesto T-cell-dependent antigens.

Accordingly, one aspect of the invention is a method for treatingnaturally occurring age-related decline in immune function, comprisingadministering to a warm blooded animal at least one steroid hormonewhich enhances T cell lymphokine production, wherein the steroid hormoneis selected from the group consisting of DHEA and DHEA cogeners.

Another aspect of the invention is a method for treating naturallyoccurring age-related decline in immune function, comprisingadministering to a warm blooded animal at least one steroid hormonewhich enhances T cell lymphokine production, wherein the steroid hormoneis a DHEA prohormone.

Yet another aspect of the invention is a method for augmenting in animmunodeficient individual an immune response comprising administeringto the individual a pharmaceutical composition comprised of a prohormoneof DHEA.

An additional aspect of the invention is a method for augmenting in animmunodeficient individual an immune response comprising administeringto the individual DHEA, and wherein the immunodeficiency is due totrauma.

Still another aspect of the invention is a method for augmenting in animmunodeficient individual an immune response to an antigen comprisingadministering to the individual a steroid selected from DHEA and a DHEAcogener, wherein the administration is such that the DHEA and antigenwill drain to the same lymph node.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the biosynthetic relationship of DHEA toother steroid hormones.

FIG. 2 is a diagram of DHEA-S.

FIG. 3A is a graph showing the dose response curve for DHEA on theproduction of IL-2 and IL-4 by activated murine T cells.

FIG. 3B is a graph showing the dose response curve for DHEA-S on theproduction of IL-2 and IL-4 by activated murine T cells.

FIG. 4A is a graph showing the effect of corticosterone and/or DHEA onthe in vitro lymph node T cell response to OVA.

FIG. 4B is a graph showing the effect of dexamethasone (DEX) and/or DHEAon the in vitro lymphokine response of OVA/2, an ovalbumin-specificcloned T cell line.

FIG. 4C is a graph showing the effect of dexamethasone (DEX) and/or DHEAon the in vitro lymphokine response of OVA/3, an ovalbumin-specificcloned T cell line.

FIG. 5 is a graph showing the results of the effect of DHEA and DHEA-Sin vivo on IL-2 and IL-4 production by spleen cells.

FIG. 6 is a graph showing the results of a pulse of DHEA in vitro onIL-2 and IL-4 production by splenocytes from mice treated withcorticosterone in vivo.

FIG. 7 is a graph showing the results of DHEA and/or corticosteroneadministered in vivo on the levels of IL-2 and IL-4 production byactivated splenocytes.

FIG. 8 is a graph showing the effect of DHEA and 1,25(OH)₂ D₃administered in vivo on lymphokine production by activated splenocytes.

FIG. 9 is a graph showing age-associated changes in lymphokineproduction by activated splenocytes.

FIG. 10 is a graph showing the effect of DHEA administered in vivo onlymphokine production in splenocytes isolated from aged and mature youngmice.

FIG. 11A is a graph showing the pattern of IL-2 produced by T cells fromaged BALB/c donor mice and younger donor mice.

FIG. 11B is a graph showing the pattern of IL-4 produced by T cells fromaged BALB/c donor mice and younger donor mice.

FIG. 11C is a graph showing the pattern of IL-5 produced by T cells fromaged BALB/c donor mice and younger donor mice.

FIG. 11D is a graph showing the pattern of γ-IFN produced by T cellsfrom aged BALB/c donor mice and younger donor mice.

FIG. 11E is a graph showing the pattern of IL-3 produced by T cells fromaged BALB/c donor mice and younger donor mice.

FIG. 11F is a graph showing the pattern of GM-CSF produced by T cellsfrom aged BALB/c donor mice and younger donor mice.

FIG. 12A is a graph showing the results of DHEA-S supplementation on thecapacity of T cells from aged mice to produce IL-2.

FIG. 12B is a graph showing the results of DHEA-S supplementation on thecapacity of T cells from aged mice to produce IL-4.

FIG. 12C is a graph showing the results of DHEA-S supplementation on thecapacity of T cells from aged mice to produce IL-5.

FIG. 12D is a graph showing the results of DHEA-S supplementation on thecapacity of T cells from aged mice to produce γ-IFN.

FIG. 12E is a graph showing the results of DHEA-S supplementation on thecapacity of T cells from aged mice to produce IL-3.

FIG. 12F is a graph showing the results of DHEA-S supplementation on thecapacity of T cells from aged mice to produce GM-CSF.

FIG. 12G is a graph showing the results of chronic DHEA-Ssupplementation on the humoral responsiveness of aged mice to an OVAchallenge.

FIG. 13A is a graph showing the results of administration of a bolus ofDHEA-S to aged mice on the ability of splenocytes from the treated miceto have restored IL-2 production.

FIG. 13B is a graph showing the results of administration of a bolus ofDHEA-S to aged mice on the ability of splenocytes from the treated miceto have restored IL-4 production.

FIG. 13C is a graph showing the results of administration of a bolus ofDHEA-S to aged mice on the ability of splenocytes from the treated miceto have restored IL-5 production.

FIG. 13D is a graph showing the results of administration of a bolus ofDHEA-S to aged mice on the ability of splenocytes from the treated miceto have restored γ-IFN production.

FIG. 13E is a graph showing the results of administration of a bolus ofDHEA-S to aged mice on the ability of splenocytes from the treated miceto have restored IL-3 production.

FIG. 13F is a graph showing the results of administration of a bolus ofDHEA-S to aged mice on the ability of splenocytes from the treated miceto have restored GM-CSF production.

FIG. 13G is a graph showing the results of administration of a bolus ofDHEA-S to aged mice on the production of anti-ovalbumin antibodies bythe treated mice.

FIG. 14 is a graph showing the effect of topical DHEA application toaged mice on the production of anti-ovalbumin antibodies.

FIG. 15A is a graph showing the effect of thermal injury on theproduction of lymphokines by activated T-cells.

FIG. 15B is a graph showing the effect of thermal injury on contacthypersensitivity reactions.

FIG. 16A is a graph showing the effect of DHEA treatment in vitro on theproduction of IL-2 by activated splenocytes from thermally injured andcontrol mice. Panel A shows the bioactivity of IL-2. Panel B shows theimmunoactivity of γIFN. Panel C shows the immunoactivity of IL-4. PanelD shows the immunoactivity of IL-5. Panel E shows the immunoactivity ofIL-3. Panel F shows the immunoactivity of GM-CSF.

FIG. 16B is a graph showing the effect of DHEA treatment in vitro on theproduction of γ-IFN by activated splenocytes from thermally injured andcontrol mice.

FIG. 16C is a graph showing the effect of DHEA treatment in vitro on theproduction of IL-4 by activated splenocytes from thermally injured andcontrol mice.

FIG. 16D is a graph showing the effect of DHEA treatment in vitro on theproduction of IL-5 by activated splenocytes from thermally injured andcontrol mice.

FIG. 16E is a graph showing the effect of DHEA treatment in vitro on theproduction of IL-3 by activated splenocytes from thermally injured andcontrol mice.

FIG. 16F is a graph showing the effect of DHEA treatment in vitro on theproduction of GM-CSF by activated splenocytes from thermally injured andcontrol mice.

FIG. 17A is a graph showing the effect of DHEA treatment in vivo on theproduction of IL-2 by activated splenocytes from thermally injured andcontrol mice. Panel A shows the bioactivity of IL-2. Panel B shows theimmunoactivity of γIFN. Panel C shows the immunoactivity of IL-4. PanelD shows the immunoactivity of IL-5. Panel E shows the immunoactivity ofIL-3. Panel F shows the immunoactivity of GM-CSF.

FIG. 17B is a graph showing the effect of DHEA treatment in vivo on theproduction of γ-IFN by activated splenocytes from thermally injured andcontrol mice.

FIG. 17C is a graph showing the effect of DHEA treatment in vivo on theproduction of IL-4 by activated splenocytes from thermally injured andcontrol mice.

FIG. 17D is a graph showing the effect of DHEA treatment in vivo on theproduction of IL-5 by activated splenocytes from thermally injured andcontrol mice.

FIG. 17E is a graph showing the effect of DHEA treatment in vivo on theproduction of IL-3 by activated splenocytes from thermally injured andcontrol mice.

FIG. 17F is a graph showing the effect of DHEA treatment in vivo on theproduction of GM-CSF by activated splenocytes from thermally injured andcontrol mice.

FIG. 18 is a graph showing the effect of DHEA treatment in vivo oncontact hypersensitivity responses of thermally injured and controlmice.

FIG. 19 is a graph showing the effect of DHEA treatment on resistance toL. monocytogenes in control and thermally injured C3H mice.

FIG. 20 is a graph showing the effect of DHEA, 16α-bromo-DHEA and16α-chloro-DHEA in vitro on IL-2 production by lymphocytes from agedmice.

FIG. 21 is a graph showing the effect of topically applied DHEA,16α-bromo-DHEA and 16α-chloro-DHEA on the anti-Ovalbumin antibodyresponse in aged mice.

DETAILED DESCRIPTION OF THE INVENTION

The most important function of the immune system is to provide its hostwith protection against diseases. To carry out these tasks, a large anddiverse array of effector mechanisms have evolved, the majority of whichexhibit antigen specificity. Each individual effector mechanismpossesses a degree of uniqueness with respect to its ability toinfluence the rate of progression, to detoxify, or to promote theelimination of microbial pathogens or tumor cells. Such a diversity inavailable mechanisms is absolutely essential since no single effectorresponse can effectively deal with all forms of pathogenic insults.Furthermore, to protect normal function of the various non-lymphoidorgan systems and tissues of the body requires careful selection,activation, and compartmentalization of the most appropriate types ofimmune effector mechanisms. Equally important is the simultaneouscapacity to down-regulate the development of other types of responses.Immunologic effector responses must, therefore, be both effective andpractical, and at the same time be appropriately regulated anatomicallyto reduce the risk of pathologic consequences.

The nonlymphoid tissues and organs of the body, which work collectivelyto sustain the life of the host, must also be capable of providingregulatory information to cells of the immune system. This information,mediated through the activities of inflammation-induced tissuecytokines, prostaglandins, plus other types of biological responsemodifiers, becomes integrated into the complex equation to control themechanisms which regulate effector response selection.

T cells, through their capacity to produce a number of lymphokines inresponse to activation, play a central role in guiding the developmentof immune effector responses. Mechanisms which operate to control thesynthesis and secretion of these pleiotropic biologic responsemodifiers, therefore, directly influence the quantitative andqualitative nature of immunity. The lymphokines and cytokines provideimportant information, not only to cells of the immune system, but alsoto cells of the other tissue and organ systems. For this information tobe meaningful, it is essential that lymphokine production remainstightly controlled at the levels of both cellular source and duration.Autocrine and paracrine effects by lymphokines and cytokines should bethe norm, since only a few species of lymphokines and cytokines arecapable of working effectively when provided via endocrine routes. Theseessential anatomic restrictions, therefore, cannot be adequatelyprovided by bolus injection of recombinant lymphokines and/or cytokines,and may explain the limited success associated with this form oftherapy.

The vast majority of the T cells in the peripheral circulation are knownto reside within the recirculating T cell pool. These cells continuouslyenter and exit secondary lymphoid organs throughout the body,maintaining residence within any particular site for only finite periodsof time. Over the lifespan of any individual mature T cell, therefore,it has probably taken up temporary residence in most of a host'ssecondary lymphoid organs. T-cell recirculation provides the immunesystem with a means for clonally-restricted T cells to provide a levelof surveillance over all the tissue and organ systems.

It is universally accepted that most T cells acquire their specificityfor antigen, and a self-MHC-restricting element, during processes whichoccur during their ontogeny within the thymus. However, the extent towhich intrathymic maturation confers genetic restrictions uponindividual T cells that regulate their potential for immunologicinvolvement has not been delineated.

A general concept which explains the results in the Examples, but whichis not intended to limit our invention, is that the genetic programs ofresting recirculating T cells are continuously being altered byextrinsic environmental influences. The steroid hormones, eitherpresented in their active forms systemically (e.g. glucocorticosteroids(GCS)), or being provided to T cells only within discretemicroenvironments as a consequence of end-organ metabolism e.g., DHEA,DHT, OR 1,25(OH)₂ D₃ ! perform important roles in this process. Thebasal regulation of the immune system at the level of the T cellrequires the continual presence of the needed substrates (prohormones).The anatomic compartmentalization of functional potential for T cells,therefore, would be dependent on the cellular source of the steroidmetabolizing enzymes able to convert the steroid hormone substrates totheir bioactive species. Our studies show that macrophages can containeach of these enzymes.

More specifically, DHEA-S is naturally converted to DHEA in theperipheral lymph nodes of animals with normal immune function. The DHEAproduced then influences the T lymphocytes within the lymph node andexerts controlling influences on their ability to respond whenactivated. This provides a means to regulate the potential of T cells byfluctuating the degree to which a particular steroid hormone existswithin a particular tissue. Old individuals and/or stressed individuals,including humans, lose the capacity to produce DHEA-S, resulting inaltered T-cell responsiveness. The invention in its various embodimentsrestores the metabolite produced from DHEA-S in the anatomic compartmentin which T-cell responsiveness is required for normal immune responsesto T-cell-dependent antigens.

As used herein, the term "individual" refers to a vertebrate andpreferably to a member of a species which exhibits DHEA-S sulfataseactivity, and includes but is not limited to domestic animals, sportsanimals, and primates, including humans.

The term "effective amount" refers to an amount of DHEA-S, DHEA, or DHEAcogener sufficient to restore normal immune responsiveness in animmunodeficient subject to which it is administered, i.e., it restoresDHEA in the anatomic compartment in which T-cell responsiveness isrequired to a level for normal immune responses to T-cell-dependentantigens. The exact amount necessary will vary from subject to subject,depending on the species, age, and general condition of the subject, theseverity of the condition being treated, the mode of administration,etc. Thus, it is not possible to specify an exact effective amount.However, the appropriate effective amount may be determined by one ofordinary skill in the art using only routine experimentation.

As used herein, the term "immunodeficient individual" means anindividual whose response to immune stimulation to a foreign antigen issignificantly less than that of the average of normal individuals of thesame species. Methods of determining "immunodeficiency" are known in theart, and include, for example, an examination of lymphokine productionby activated T cells; the ability of the individual to demonstratecontact hypersensitivity; the ability of the individual to raise ahumoral response to antigen challenge, or the resistance of theindividual to infection by microorganisms.

"Treatment" refers to the administration of a composition to anindividual which yields a protective immune response, and includesprophylaxis and/or therapy.

An "antigen" refers to a molecule containing one or more epitopes thatwill stimulate a host's immune system to make a secretory, humoraland/or cellular antigen-specific response. The term is also usedinterchangeably with "immunogen".

An "immunological response" to a composition or vaccine comprised of anantigen is the development in the host of a cellular and/orantibody-mediated immune response to the composition or vaccine ofinterest. Usually, such a response consists of the subject producingantibodies, B cells, helper T cells, suppressor T cells, and/orcytotoxic T cells directed specifically to an antigen or antigensincluded in the composition or vaccine of interest.

By "vaccine composition" or "vaccine" is meant an agent used tostimulate the immune system of an individual so that current harm isalleviated, or protection against future harm is provided.

"Immunization" refers to the process of inducing a continuing high levelof antibody and/or cellular immune response which is directed against anantigen to which the organism has been previously exposed.

As used herein, the term "prohormone" pertains to water solubleprecursors of DHEA, i.e., DHEA derivatives from which DHEA may besynthesized in vivo, for example, DHEA-S (and other precursors known inthe art).

As used herein, a "pharmacologic dose" is one which gives a desiredphysiological effect.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,biochemistry, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See, e.g., ANIMALCELL CULTURE (R.I. Freshney ed. 1986); IMMOBILIZED CELLS AND ENZYMES(IRL Press, 1986; the series, METHODS IN ENZYMOLOGY (Academic Press,Inc.), IMMUNOCHEMICAL METHODS IN CELL AND MOLECULAR BIOLOGY (AcademicPress, London), Scopes, (1987); and HANDBOOK OF EXPERIMENTAL IMMUNOLOGY,Volumes I-IV, (D. M. Weir and C. C. Blackwell, eds., 1986.) All patents,patent applications, and publications mentioned herein, both supra andinfra, are hereby incorporated herein by reference.

One embodiment of the invention is a method for enhancing or maximizingthe production of T cell lymphokines which are correlated withprotective immunity. The method comprises exposing T cell lymphocyteswhich have a potential to make selected T cell lymphokines toappropriate concentrations of particular steroid hormones prior toactivation. If the exposure is in vitro, the particular steroid hormoneto which the T cell lymphocyte is exposed depends upon the lymphokinewhich is selected for enhancement or maximized production. If theexposure is in vivo, a pharmaceutical composition comprised of thesteroid hormone is administered to the individual, particularly to animmunodeficient individual. Immunodeficiency may be for a variety ofreasons, for example, age, i.e., very young (e.g., neonate) or aged,stress, or trauma. The administration to the individual is by techniquesknown in the art, including, for example, parenteral, transdermal, ortransmucosal.

In accordance with the invention, exposing T cell lymphocytes which havea potential to make selected T cell lymphokines to DHEA or a DHEAcogener prior to activation enhances the production of IL-2, IL-3,γ-IFN, and GM-CSF. DHEA cogeners which are useful in the invention havethe following structure: ##STR1## in which R is hydrogen in alpha orbeta configuration or nothing, resulting in a double bond between carbonatoms 5 and 6; R₁ is hydrogen or a halogen in α-configuration; and R2 isoxygen or methyl ketone (--COCH₃); or ##STR2## in which R is hydrogen inalpha or beta configuration or nothing, resulting in a double bondbetween carbon atoms 5 and 6; R₁ is hydrogen or a halogen inα-configuration; R2 is oxygen or methyl ketone (--COCH₃); and R₃ is OHor sulfate.

Alternatively, and particularly in vivo, the selected steroid hormonesmay be administered to individuals through precursor substances whichare then metabolized to DHEA or its metabolites. For example, thesulfonated form of DHEA, DHEA-S, can be administered provided that theadministration is to an individual that can metabolize the prohormone toDHEA by tissue-associated DHEA-sulfatases.

The simultaneous enhancement or maximization of the production of morethan one T cell lymphokine may be achieved by exposing the T celllymphocyte to more than one steroid hormone prior to activation. Theexposure to more than one steroid hormone can be simultaneous orsequential. The concentration of each of the steroid hormones should bebalanced to achieve the desired enhancing effects. For example, if itwere desirous to enhance the production of IL-2, γ-IFN, and IL-4, the Tcell lymphocytes could be exposed to physiologic or pharmacologic levelsof DHEA and a physiologic level of GCS. This would avoid the IL-2 andγ-IFN depression which is characteristic of a pharmacologic level ofGCS.

Evidence derived from experimental and clinical observations indicatesthat immunologic reactions elicited to either simple or complex antigensoften manifest as a balanced heterogenous blend of both cellular andhumoral components, with the fractional contribution of any individualtype of effector mechanism oftentimes dominating the overall response.This level of heterogeneity is essential to the development of aprotective immune response. Alterations to this natural balance, whethercaused by genetic or physiologic changes associated with age or stressor trauma, can lead to a depressed capacity to elicit protective immuneresponses, and might also lead to immunologic responses havingpathologic consequences.

Administration of steroid hormones, particularly DHEA-S or DHEA orDHEA-cogeners in accordance with the invention would be useful intreating such immune system imbalances in individuals. For example,immunosuppression (a form of immunodeficiency) in warm blooded animalsmay be mediated by elevated GCS levels. These elevated levels can resultfrom a variety of causes including, but not limited to, stress andtrauma (including, for example, post-surgical trauma, burn trauma), as asecondary consequence to any clinical condition which causes an elevatedproduction of IL-1, or therapeutic treatment for a variety of clinicalconditions. The elevated GCS levels can result in an imbalance in theproduction of essential interleukins. The normal balance of essentialinterleukin production may be restored by therapeutic administration ofDHEA, DHEA-S, or its cogeners.

Additionally, if it were known that elevated GCS levels were the resultof certain behavior or maladies, administration of DHEA-S, DHEA, or DHEAcogeners could be used as a prophylaxis prior to the onset of theelevation in GCS levels and resultant immunosuppression. In these cases,where the administration is chronic, it is advisable to use theprohormone form (e.g., DHEA-S) to prevent side effects associated withthe administration of large doses of DHEA. For instance, there is abovine malady commonly known as "Shipping Fever" which has a high rateof morbidity and mortality associated with the stress induced by longdistance shipment. This stress is associated with chronic increasedlevels of GCS. Prophylactic administration of prohormones (e.g., DHEA-S)in accordance with the invention prior to and/or during bovine shipmentmay counteract the immunosuppressive effects of the chronically elevatedGCS levels, reducing the risk of these animals to infectious agents andweight loss.

The invention may also be used as a diagnostic tool in evaluatinglymphokine production deficiency. In this application T cell lymphokineproduction of a first group of T-cell lymphocytes which have a potentialto make selected T cell lymphokines after T cell lymphocyte activationis measured. A second group of the same type of T cell lymphocytes isexposed to a particular steroid hormone prior to T cell activation. Theselected T cell lymphokine is then measured after activation. The amountof T cell lymphokine production of the two groups of T cell lymphokinesare compared. The sensitivity of the diagnostic tool is maximized whenthe amount of the particular steroid hormone to which the second groupof T cell lymphocytes is exposed is sufficient to maximize theproduction of the T cell lymphokines which the particular steroidhormone enhances. For example, if the T cell lymphokine is IL-2, orIL-3, or γ-IFN, or GM-CSF, the preferred steroid hormone may be selectedfrom the group DHEA or a DHEA cogener having the structure recitedabove.

Another application of the invention is to treat naturally occurringage-related decreases in immune function, which correlate with adecrease in circulating DHEA-S levels. Associated with age relateddecline in immune function is a decrease in the production of certainlymphokines. Treatment of aging, warm blooded animals with steroidhormones, preferably DHEA-S or DHEA-cogeners, substantially restores theproduction of the selected lymphokines involved in the cascade leadingto immunologic competence.

Generally, the person in charge of the administration of the steroids,DHEA, DHEA-S, or DHEA-cogeners will choose the appropriate form of thesteroid based upon the compartmentalization effect and metabolicproducts resulting therefrom. For example, if the indication foradministration is prophylaxis or chronic therapeutic treatment, theprohormone DHEA-S is preferred to escape the side effects associatedwith of the administration of chronic high levels of DHEA. In this casethe level of DHEA-S may be in the range of about 5 to about 100 mg perday, preferably may be in the range of about 10 to about 80 mg per day,and even more preferably may be in the range of about 15 to about 60 mgper day.

Alternatively, if the indication for treatment is acute trauma orstress, it may be preferable to treat with a bolus administration ofDHEA. The bolus administration may be in the range of about 1 to about20 mg per kg of body weight, more usually may be in the range of about 2to about 10 mg per kg of body weight, and preferably may be in the rangeof about 3 to about 8 mg per kg of body weight.

The compounds of the present invention can be administered to theimmunologically deficient individual in a variety of forms adapted tothe chosen route of administration, for example, orally, intravenously,intramuscularly, or via subcutaneous, topical, or inhalation routes.

Pharmaceutical compositions made up of formulations comprised of thesteroids (particularly DHEA, DHEA-S, and DHEA-cogeners) and suitable forthe administration by each of these routes may be prepared by one ofordinary skill in the art. See, for example, Remington's PharmaceuticalSciences, 17th Edition (1985, Mack Publishing Company, Easton, Penn.).For example, the pharmaceutical composition containing the steroid mayalso contain a carrier or solid or be encapsulated in a material that isnon-toxic to the inoculated animal and is compatible with the steroid.Suitable pharmaceutical carriers include liquid carriers, such as normalsaline and other non-toxic salts at or near physiologicalconcentrations, and solid carriers not used for humans, such as talc orsucrose, also feed for farm animals. When used for administering via thebronchial tubes, the steroid hormone is preferably presented in the formof an aerosol.

Another application of the invention is to use a pharmaceuticalcomposition containing the steroid, preferably DHEA or a DHEA cogener,as a vaccine adjuvant to augment or selectively direct thevaccine-induced immune response down a protective immunologic pathway.When the individuals are immunized with an immunizing agent,administration of the steroid may be prior to or contemporaneously withthe vaccination. Typical methods of administering the steroid hormoneinclude implants, mixing the steroid hormone with the immunizing agent,or topically applying the steroid hormone composition to skin siteswhich drain to the same lymph nodes as the antigen of the vaccine. Thislatter method is preferably used with individuals who areimmunologically deficient due to low levels of DHEA-S and/or DHEA and inwhom one wishes to augment the immune response, for example, the aged orneonates or individuals who are therapeutically immunosuppressed.

Described below are examples of the present invention which are providedonly for illustrative purposes, and not to limit the scope of thepresent invention. In light of the present disclosure, numerousembodiments within the scope of the claims will be apparent to those ofordinary skill in the art.

EXAMPLES Example 1 DHEA enhances IL-2 Production by Activated Murine TCells

In this experiment the capacity of DHEA and DHEA-S to alter theproduction of IL-2 and IL-4 following in vitro lymphocyte treatment orexposure was evaluated. DHEA significantly enhanced the production ofIL-2 over a wide dose range, and DHEA-S, over the same dose range, hadno effect on IL-2 and IL-4 production. FIG. 3A is the dose responsecurve of DHEA and FIG. 3B is the dose response curve of DHEA-S developedin this experiment.

Spleen cells obtained from normal BALB/c mice were prepared as a singlecell suspension at a concentration of 1×10⁷ cells/ml in RPMI 1640supplemented with 2 mM L-glutamine, 5×10⁻⁵ M 2-mercaptoethanol, 20 μg/mlgentamycin-sulfate, and 1% nutridoma-NS (Boehringer-Mannheim).Individual aliquots of cells were then pulsed for 30 minutes at 37° C.with the indicated concentrations of DHEA or DHEA-S. After pulsing, thecells were washed several times in balanced salt solution, resuspendedin fresh medium, and then dispensed into 24-well culture plates with astimulatory concentration of anti-CD3 (Leo et al. (1987), Proc. Natl.Acad. Sci. USA 84:1374). After a 24-hour incubation period, culturesupernatants were harvested for assessment of IL-2 and IL-4 activityusing the method of Mossman (J. Immunol. Meth. (1983)). In thisexperiment, 100% control titers of IL-2 and IL-4 from normal stimulatedsplenocytes in FIG. 3A were 640 and 160 units/ml, respectively. Forcontrol splenocytes from FIG. 3B, 100% control titers of IL-2 and IL-4were 2560 and 320 units/ml, respectively.

This same experiment was repeated to assay for γ-IFN production. A doseresponse curve similar to that reported in FIG. 3A for DHEA was obtainedfor γ-IFN.

This same experiment was performed using the DHEA cogener 16-alpha-bromoDHEA in place of DHEA. A dose response curve similar to that reported inFIG. 3A was obtained for 16-alpha-bromo DHEA.

Example 2 DHEA enhances IL-2 Production in GCS-Treated NormalSplenocytes and Cloned T Cell Lines

The capacity of DHEA to facilitate a reversal of glucocorticoid-inducedsuppression of IL-2 production by either normal murine lymphocytes, orcloned T cell lines with similarities to either Th1-type or Th2-typehelper T cells was evaluated.

Single cell suspensions of normal murine spleen cells were prepared inNutridoma-supplemented complete RPMI at 10⁷ cells/ml. They were thenpulsed with 10⁻⁷ M corticosterone and/or 10⁻⁸ M DHEA as described inFIG. 4A. After several washes, the cells were stimulated with anti-CD3.The enhancement of IL-2 production by DHEA exposed normal splenocytes isshown in FIG. 4A. FIG. 4B and FIG. 4C show the regulation of lymphokineproduction by two ovalbumin (OVA)-specific cloned T cell lines. TheOVA-specific T cell clones were derived from nylon-wool enriched splenicT cells from OVA-immunized (C3H×C57/B6) F₁ mice using the method ofBerzofsky (1985), J. Immunol. 35:2628. OVA/3 and OVA/2 cell lines werederived from different clonings, each having distinct patterns oflymphokine production. Culture conditions and assay procedures for IL-2and IL-4 are as in Example 1.

Referring to FIG. 4A, exposure of splenocytes to the effects ofcorticosterone (10⁻⁷ M) greatly reduced the capacity of cells to produceIL-2 subsequent to activation with anti-CD3. DHEA treatment aloneaugmented IL-2 production. Lymphocytes exposed to corticosterone andDHEA, followed by their activation in vitro, produced normal or enhancedlevels of IL-2 and enhanced levels of IL-4.

Referring to FIG. 4B and FIG. 4C, OVA/2 an ovalbumin (OVA)-specificcloned T cell line with characteristics similar to Th2-type cells) andOVA/3 (a cloned T cell line with characteristics similar to Th1-typecells), were exposed in vitro to the effects of DHEA and/orglucocorticoids prior to their culture with antigen and syngeneicantigen-presenting cells. As shown in FIG. 4C, DHEA treatment of OVA/3greatly augmented the capacity of this cell line to produce IL-2, whileexposure to DEX resulted in an IL-4 dominant phenotype, similar to whatis observed with Th2-type clones. Treatment of OVA/3 with DEX followedby DHEA, resulted in a marked elevation in IL-2 production with only aminimal enhancement of IL-4. As shown in FIG. 4B, the effects of steroidtreatment on the capacity of OVA/2 to produce TCGF gave comparableresults. DHEA exposure of this T cell clone was capable of shifting thepattern of TCGF production from a Th2-like to a Th1-like phenotype (IL-2dominant), while DEX treatment alone augmented IL-4 production followingactivation in vitro with OVA. Treatment of OVA/2 with both DEX and DHEAcaused an enhanced capacity to produce both IL-2 and IL-4.

Example 3 A Single Injection of Mice with DHEA or DHEAS Enhanced theBiosynthesis of IL-2 by Activated Lymphoid Cells

This example demonstrates the effects of in vivo administration of DHEAand DHEA-S on IL-2 and IL-4 biosynthesis.

Groups of (C3H×BL/6)F1 mice were given a single intraperitonealinjection of 100 μg DHEA or DHEA-S. After three days, spleen cells fromthe treated groups, plus spleen cells from an untreated age-matchedcontrol group, were prepared for culture as described in Example 1. Therelative titers of IL-2 and IL-4 in the 24-hour culture supernatantswere determined in the presence of anti-IL-2, or anti-IL-4, or bothanti-IL-2 and anti-IL-4, or no blocking antibodies. The assay was readvisually. FIG. 5 presents the results of the study. Non-activatedcultured lymphoid cells produced undetectable (less than 2 units) ofeither IL-2 or IL-4.

Example 4 DHEA Enhances IL-2 Production in Splenocytes fromCorticosterone-Treated Mice

The reversal of the inhibitory effects caused by chronic glucocorticoidadministration to normal mice in vivo on the capacity of their T cellsto produce IL-2 was demonstrated as follows.

Biodegradable pellets (Innovative Research, Inc.) containingcorticosterone and designed to deliver the steroid at a dose of 5 μg/hrwere implanted subcutaneously into (C3H×BL/6) F₁ mice. The splenocytesfrom the mice were harvested 72 hours after the implantation. Prior toactivation, the splenocytes were pulsed with a short pulse of DHEA (10⁻⁸M). Culture and assay procedures for IL-2 and IL-4 were as described inExample 1. The results are presented in FIG. 6.

As seen in the figure, the DHEA pulse caused a significant enhancementof IL-2 production. Under these conditions, the glucocorticoid-inducedaugmentation in IL-4 synthesis was not affected, resulting in apopulation of lymphoid cells capable of producing high levels of bothIL-2 and IL-4.

Example 5 DHEA in vivo Enhances IL-2 Production in Mice with and withoutCorticosterone Treatment

This example demonstrates that DHEA administered in vivo influences theprofile of T cell growth factors (TCGF) produced by splenocytes isolatedfrom treated animals.

Biodegradable pellets (Innovative Research, Inc.) containingcorticosterone or DHEA that deliver the steroids at 5 μg/hr wereimplanted subcutaneously into three separate groups of BALB/c mice 72hours prior to harvesting and preparation of spleen cells. Single cellpreparations of splenocytes from each group were cultured as describedin Example 1, and stimulated with the polyclonal T cell mitogen,anti-CD3. After 24 hours, culture supernatants were collected andassayed for IL-2 and IL-4 activity as described in Example 1.

As seen in FIG. 7, the stimulation of splenocytes isolated from normalanimals consistently gave a standard pattern of TCGF production whereIL-2 dominated over IL-4. Lymphocytes isolated fromcorticosterone-treated animals demonstrated a marked reversal of thispattern; IL-4 consistently represented the dominant TCGF. Similar towhat is observed following an in vitro treatment with this androgensteroid, activated splenocytes from the DHEA-treated animals exhibitedan enhancement in IL-2 production. Under conditions where both steroidswere elevated in vivo, it was found that isolated splenocytes from theseanimals produced enhanced levels of both IL-2 and IL-4 subsequent totheir activation with anti-CD3 in vitro.

Example 6 The Effect of DHEA and 1,25-dihydroxyvitamin D3 in vivo onIL-2 and IL-4 Production in vitro

CH3 mice received implants of biodegradable DHEA or 1,25(OH)₂ D₃ pelletsdesigned to deliver steroid at a rate of 5 and 1.25 μg/hr, respectively.Three days after implantation, both the steroid treated groups and anormal control group of mice were immunized in the hind footpads with100 μg OVA in CFA. Ten days after immunization, the draining lymph nodesand spleens from all groups were prepared for culture. Lymph node cellswere stimulated with 100 μg OVA. Culture supernatants were assayed forIL-2 and IL-4 activity after 24 hours using the HT-2 bioassay. Theresults are shown in FIG. 8. From the figure it may be seen that DHEAadministration caused approximately a four-fold increase in IL-2production, and no stimulation of IL-4 production. In contrast,1,25(OH)₂ D₃ administration caused an approximate eight-fold increase inIL-4 production, but did not stimulate IL-2 production.

Similar alterations in the ability of antigen-activated T cells toproduce IL-2 and IL-4 were observed when the steroid hormone was mixedwith the immunizing antigen, or was topically applied to skin sitesabove the site of vaccination.

Example 7 The Reversal of Age-related Decline in IL-2 and γ-IFNProduction

This example demonstrates age-related decline in the production ofcertain lymphokines, and restoration by steroid hormone treatment. Thelymphokines assayed are IL-2, IL-4, and γ-IFN; the steroid hormoneadministered is DHEA.

Age associated changes in lymphokine production are shown in FIG. 9.(CH3×BL/6)F₁ mice of the indicated ages were sacrificed and their spleencells prepared for culture with mitogen, anti-CD3. Culture supernatantswere harvested and evaluated for the relative contribution of IL-2 andIL-4 using the HT-2 bioassay. As seen in the figure, aged mice (13 and17 months) produced significantly less IL-2 and significantly more IL-4than did younger mature mice (3 and 7 months). Non-activated cellsproduced less than 1 unit of either IL-2 or IL-4.

A reversal of the aging effect on IL-2 production by DHEA is shown inFIG. 10. In the study, both young (6 mos.) and old (16 mos.) mice wereimplanted with DHEA pellets delivering a dose of 5 μg/hr. After threedays, DHEA groups and control age-matched groups were sacrificed andtheir spleen cells prepared for culture with the mitogen anti-CD3.Culture supernatants were harvested and evaluated for the relativecontribution of IL-2 and IL-4 using the HT-2 bioassay, and for γ-IFNusing the assay of Green. Non-activated cells produce less than 1 unitof either IL-2 or IL-4 and no detectable γ-IFN.

Similar enhancement in the capacity of lymphocytes derived from old miceto produce IL-2 was observed following a direct exposure of thesplenocytes in vitro to DHEA (10⁻⁹ to 10⁻⁷ M).

Example 8 Activated T cells from Aged donors Produce an Altered Patternof Lymphokines Compared to Normal

Using a serum-free culture system which allows in vitro activation oflymphocytes under conditions devoid of the restrictive regulatoryinfluences by platelet-derived growth factor and other serum-associatedmodulators of cellular activity, lymphocytes from mature adult and agedmice were compared for lymphokine production following T-cellactivation. Splenocyte cultures were either stimulated with 1 μg/mlanti-CD3ε or left unstimulated to control for any spontaneous lymphokineproduction. After a 24-hour incubation period, cell-free culturesupernatants were analyzed for lymphokine content. The materials andmethods used in these studies were as follows.

The BALB/c mice used were bred from breeding stock originally purchasedfrom the National Cancer Institute. The source of aged mice for theseexperiments was retired breeders from our own colony. Age andsex-matched mice, ranging in age from 13 to 39 weeks for mature adult,and 112-120 weeks for aged mice were used.

Monoclonal antibody reagents were prepared from culture supernatants ofB-cell hybridomas adapted to growth under serum-free conditions. Thehybridoma clones secreting rat anti-murine γ-IFN (XMG1.2), AND RATANTI-MURINEil-5 (TRFK4 and TRFK5) were obtained from DNAX (Palo Alto,Calif.). The hybridoma clone producing hamster anti-murine CD3εmonoclonal antibody, 1452C-11.2, was obtained from J. Bluestore(University of Chicago). The hybridoma producing antibody specific formurine IL-4 (11B11) and murine γ-IFN (R46A2) was purchased from theATCC. A number of purified rat anti-murine cytokine antibodies werepurchased from PharMingen (San Diego, Calif.) and used for quantitationof specific murine cytokines by capture ELISA; these were anti-murineIL-3 antibodies (cat. nos 18011D and 18022D), biotinylated anti-IL-4(cat. no. 18042D), anti-murine GM-CSF antibodies (cat. nos. 18091D and18102D).

Murine recombinant γ-IFN was obtained from Genentech (5×10⁶ units/mgprotein) and used as a reference in the γ-IFN bioassays. Murinerecombinant IL-2, IL-4 and IL-5, were derived from culture supernatantsof X63Ag8-653 cells transfected with multiple copies of a single murineinterleukin gene. After the relative concentration of each lymphokine inculture was determined by a comparison to a known recombinant standard,these reagents were used as reference lymphokines in both bioassays andcapture ELISA. Other sources of purified, murine, reference lymphokineswere IL-5 obtained as a gift from R. Coffman, DNAX, or IL-2 and IL-4purchased from Collaborative Research Inc. (Bedford, Mass.). Ovalbumin(Sigma Chemicals, St. Louis, Mo.) was dissolved in double distilledwater at a concentration of 20 mg/ml. The solution was filter sterilizedand frozen in 3 ml aliquots at -20° C. For immunization, ovalbumin wasmixed with the commercial aluminum hydroxide preparation (Maalox). Onehundred μg in 25 microliters Maalox was injected into a single hindfootpad.

Single cell suspensions of lymphoid cells were prepared from appropriatelymphoid organs of normal mice, washed twice in sterile balanced saltsolution and cultured at a density of 1×10⁷ cell/ml/well with a T-cellspecific mitogen, routinely anti-CD3ε, in a 24-well Cluster cultureplate (Costar, Cambridge, Mass.) for a period of 24 hours to elicitlymphokine secretion. Cell-free culture supernatants were collected andstored at -20° C. until assayed for lymphokine content. The cultureperiod, cell concentrations, and culture medium, consisting of RPMI 1640supplemented with 1% Nutridoma-SR (Boehringer-Mannheim), antibiotics,200 mM L-glutamine and 5×10⁻⁵ M 2-mercaptoethanol, were all carefullyevaluated to determine the optimal conditions for stimulating productionof the lymphokines under evaluation.

HT-2 cells were used as an indicator cell line for the bioassay of IL-2,using a modification of a colorimetric assay for cell viability. Eachtest supernatant is titrated in duplicate in Nutridoma-SR-supplementedmedia (referred to as serum-free) containing 4×10³ HT-2, and saturatingamounts of anti-IL-4 monoclonal antibody. During the final 4 hours of a24-hour incubation, 5 μg of 3- 4,5-Dimethylthiazole-2-yl!-2,5 diphenyltetrazolium bromide (MTT) is added to each culture, followed by theaddition of 100 microliters of a 20% SDS/50% dimethylformamide solutionto dissolve formazan crystals. Spectrophotometric readings are recordedat 570 nm-650 nm. One unit of activity in a test supernatant isequivalent to the O.D. of a half-maximal response of HT-2, relative to astandard recombinant source.

Where indicated, the amount of other cytokines in test supernatants wasquantitated by capture ELISA, adapted from the method of Schumacher.Briefly, 100 microliters of 2 μg/ml capture antibody in 0.05M Tris-HCV(pH9.6) was adsorbed to the wells of a 96-well microtest plate, washedand blocked with PBS/1% BSA. Test supernatants and 2-fold serialdilutions of the appropriate reference cytokine (100 microliters/well)were dispensed and after sufficient incubation and washing, 100microliters of biotinylated-detection antibody, 1 μg/ml, was dispensedinto each well. The ELISA was developed using avidin-HRP andABTS-substrate. Spectrophotometric readings were recorded at 405 nM. Thelimit of detection for most of these cytokines is 15-30 pg/ml.

Anti-Ovalbumin antibody ELISA was performed as follows. Ovalbumin (SigmaChemical, St. Louis, Mo.) was diluted to a concentration of 20 μg/ml in50 mM Tris-HCl (pH 9.6). 100 microliters/well of this solution was usedto coat the wells of high protein binding, microtiter plates (Corningcat. no. 2581) following an overnight incubation at 4° C. The plateswere then blocked with 250 microliters PBS/10% FCS for 90 minutes at 37°C. and then rinsed multiple times with PBS/0.5% Tween 20. Serum testsamples plus positive and negative control serum antibodies weretitrated against PBS/10% FCS over eight 2-fold dilutions. After another90 minute incubation at 37° C. and multiple washes in PBS/0.5% Tween 20,100 microliters of a 1:1000 dilution of HRPO-coupled goat anti-murineIgM and goat anti-murine IgG was dispensed into each well. This step wasfollowed by an incubation at 37° C. for 90 minutes, PBS/0.5% Tween 20washes, and addition of 100 microliters of an ABTS substrate forspectrophotometric detection of antibody activity in the assay. Readingsfrom a spectrophotometer were recorded at 405 nM. The titer of specificantibody in a test serum was assigned as the inverse of the antibodydilution that was equivalent to a half-maximal response. Antibodyactivity of most sera was saturating at the lowest dilutions, implying ahigh level of efficiency in the capture and the detection ofovalbumin-specific immunoglobulin.

FIGS. 11A-FIG. 11F are graphs showing the pattern of lymphokinesproduced by T cells from aged BALB/c donor mice and younger donor mice.In the study, splenocytes were prepared from groups of 3 mature adult(28 weeks of age) and 3 aged (112 weeks of age) BALB/c donor mice. 1×10⁷splenocytes were cultured under serum-free conditions in triplicate andactivated with 1 μg/ml CD3ε. Culture supernatants were analyzed for thelevel of IL-2 by quantitative bioassay, and for IL-4, IL-5, γ-IFN, IL-3and GM-CSF as described above. In the figure, bars represent the mean±SD for the value of each lymphokine presented.

As seen in FIGS. 11A-11F, the in vitro activation of splenocytes fromaged mice under serum-free conditions resulted in a reduced productionof some lymphokines and an enhanced production of others, compared tothe pattern of lymphokines produced by activated T cells from matureadult mice. Activation-induced production of IL-2, IL-3, and GM-CSF wereall significantly reduced in cell cultures from old donors, while thelevels of IL-4, IL-5, and γ-IFN were increased above normal adultlevels.

A comparison of lymphokine profiles between mature adult and aged micehas been performed using three strains of mice (BALB/c, C57BL/6 andC3H/HeN). The response of each of these strains was analyzed numeroustimes, and yielded similar results.

Example 9 Preservation of Normal Potential to Produce T-cell Lymphokinesand Generate Humoral Immune Responses by Supplementation with DHEASulfate

Circulating levels of DHEA sulfate declines markedly with advancing agein humans and other mammals. As shown above, direct treatment of T cellsfrom aged or normal murine donors with DHEA prior to activation in vitroaugmented their capacity to produce IL-2. In contrast, DHEA-S, theprohormone form of DHEA found principally in the circulation, was shownto have no direct effect on T-cell production of this lymphokine. WhenDHEA-S was administered to normal mature adults in vivo, it enhanced thepotential for IL-2 production by T cells isolated from lymphoid organshaving the greatest DHEA-sulfatose activity. The most active lymphoidorgans are those having anatomic positions downstream from nonmucosaltissues. This example demonstrates that DHEA-S supplementation in vivocan influence the age-related changes in lymphokine production andhumoral immune responses.

Groups of adult BALB/c mice, between 35 and 39 weeks of age, wereseparated into two groups. One group was provided with 100 μg/ml DHEA-Sin their drinking water. The hormone was offered ad libitum to theseanimals. The other group was left untreated. Mice were maintained onoral DHEA-S supplementation until age 114 weeks when they weresacrificed and their spleens individually analyzed for the capacity toproduce lymphokines following anti-CD3ε activation. The DHEA-S treatedand untreated mice were evaluated by comparing their responses to thelymphokine profile produced by similarly activated splenocytes frommature adult mice (13 weeks of age).

More specifically, splenocytes were prepared from the following groupsof BALB/c mice; 2 mature adult (13 weeks of age), 2 aged (114 weeks ofage), and 2 aged (114 weeks) receiving 100 μg/ml DHEA-S in theirdrinking water for the previous 61 weeks. 1×10⁷ splenocytes werecultured under serum-free conditions in triplicate and activated with 1μg/ml CD3ε. Culture supernatants were analyzed for the level of IL-2 bya quantitative bioassay, and for IL-4, IL-5, γ-IFN, IL-3 and GM-CSF bycapture ELISA.

FIGS. 12A-12F are graphs showing the results of DHEA-S supplementationon the capacity of T cells to produce a variety of lymphokines. In theFIGS. 12A-12F, bars represent the mean ±SD for the value of eachlymphokine presented. It may be seen from FIGS. 12A-12F that DHEA-Ssupplementation, administered prior to the onset of age-induced declinein immunocompetence, is accompanied by the preservation of normallymphokine production and development of normal humoral immuneresponses. DHEA-S supplementation was not only able to preserve normallevels if IL-2, IL-3, and GM-CSF production by activated T cells, butwas also able to prevent the age-related increase in γ-IFN, IL-4, andIL-5 production seen in the cell supernatants from untreated ageddonors. The results of this study demonstrate that a strikingcorrelation exists between the age-related decline in endogenous DHEAproduction (plus its metabolites), and the age-associated alterations inT-cell production of lymphokines.

The effect of DHEA-S supplementation on T cell function was alsoperformed using BALB/c, C57BL/6 and C3H/HeN strains of mice. In eachtest of this experimental approach, lymphokine production by T cellsfrom the treated aged donors had been preserved.

In order to examine the effect of DHEA-S supplementation on the abilityof old animals to mount immunologic responses to challenge with foreignprotein antigens, the following procedure was used. Groups of 5 matureadult mice (13 weeks of age), 5 aged mice (114 weeks), and 5 aged mice(114 weeks) provided with chronic DHEA-S supplementation (100 μg/mlDHEA-S in their drinking water for the previous 61 weeks, initiated at 8months of age), were footpad immunized with ovalbumin. The immunizationwas with 100 μg ovalbumin in a 25 μl volume of Maalox, administered inthe hind footpads. All animals were bled on days 0, 3, 5, 7, 10, and 14post immunization, and individual serum samples analyzed for ovalbuminspecific antibody titers by quantitative ELISA, using ovalbumin forcapture and HRPO-coupled, goat anti-murine Ig detecting antibodies withspecificity for IgM and IgG subclasses. Each ELISA assay was controlledwith sera known to be positive or negative for anti-ovalbumin activity.The titer is the inverse of the antibody dilution equal to thehalf-maximal point on the titration curve. The results of the study,shown in the graph in FIG. 12G, demonstrate that old animals providedwith chronic DHEA-S supplementation remain fully capable of rapidlymounting a significant humoral immune response to ovalbuminimmunization, with kinetics, titers, and isotype profiles (data notshown), that are almost identical to mature adult controls. As expected,the untreated aged mice responded poorly to a similar antigen challenge,producing predominantly IgM.

Example 10 DHEA-S Administration to Aged Mice Can Reverse Age-AssociatedChanges in T-cell Lymphokine Production and Their Depressed HumoralImmune Responses to Protein Antigens

As shown above, a direct exposure of lymphocytes from aged donors toDHEA in vitro, immediately altered the pattern of lymphokines producedfollowing activation. In addition, we have found that nonmucosal tissuedraining lymphoid organs possesses a far greater amount of DHEAsulfatase activity than mucosal tissue draining lymphoid organs. Thesefindings led to the hypothesis that DHEA may be serving as an effectorof positional information for lymphocytes residing in certain lymphoidcompartments. Any changes in immune function caused by the depressedproduction of substrate DHEA-S might, therefore, be reversible if DHEA-Sis reintroduced in situ. This was examined in the following studies.

Splenocytes were isolated from equal sized groups of mature adult mice(25 weeks of age), aged mice (120 weeks of age), and aged mice given asubcutaneous injection of DHEA-S (100 μg in 100 μl propylene glycol) 24hours previously. 1×10⁷ splenocytes were cultured under serum-freeconditions in triplicate and activated with 1 μg/ml anti-CD3ε. Twentyfour hours later, culture supernatants from individual cell cultureswere analyzed for the level of IL-2 by a quantitative bioassay, and forIL-4, IL-5, γ-IFN, IL-3 and GM-CSF by capture ELISA. The results, shownin FIGS. 13A-13F, demonstrate that acute replacement therapy with DHEA-Sto aged mice restores near normal patterns of T-cell lymphokines within1 day of treatment. These results strongly suggest that lymphoid cellsfrom old animals exhibit no intrinsic defects. Rather, some of the bestdocumented functional changes to the immune system which accompany agingmay be due to the reduced capacity to produce DHEA-S.

A representative study showing that the administration of a bolus ofDHEA-S to aged BALB/c mice restored the capacity of the mice to develophumoral responses is shown in FIG. 13G. In the study, groups of 5 matureadult (25 weeks of age), 5 aged (120 weeks of age), and 5 aged (120weeks) receiving 100 μg DHEA-S in 100 μl propylene glycol bysubcutaneous injection the previous 24 hours were immunized with 100 μgovalbumin in a 25 μl volume of Maalox, administered in the hindfootpads. Sera from individual mice were collected on days 0, 3, 5, 7,10 and 14 following primary immunization. The titer of anti-ovalbuminantibody was assessed by ELISA using ovalbumin for capture andHRPO-coupled, goat anti-murine Ig detecting antibodies with specificityfor IgM and IgG subclasses. Each ELISA assay was controlled with seraknown to be positive or negative for anti-ovalbumin activity.

The results in FIG. 13G show that old animals provided with DHEA-S only24 hours prior to immunization with a foreign protein antigen respondedeven better than normal mature adults in the production of antibody.

This method of reversing age-related decline in humoral responses hasbeen evaluated twice using BALB/c mice and once with C3H/HeN strains ofmice. Similar enhancements in antibody production were achieved in allgroups of DHEA-S treated, aged groups of mice.

The results discussed above support the concept that some of theage-associated changes in immune function are extrinsic in cause, andare mediated by the loss in endogenous production of an essentialregulatory steroid prohormone.

Example 11 Topical Application of DHEA to Aged Animals FacilitatesChanges in the Draining Lymph Node Microenvironment That are Conduciveto Successful Immunization

Groups of 5 mature adult (13 weeks of age) and 10 aged BALB/c mice (114weeks of age) were used in the study. All of the aged BALB/c micereceived a topical application of 10 μg DHEA in 3.5 μl 95% ethanol tothe right hind footpad, 3 hours prior to immunization with 100 μgovalbumin in a 25 μl volume of Maalox. Five of the aged mice wereimmunized in the right hind footpad (site identical to the steroidapplication), and the other 5 immunized in the left hind footpad (siteopposite to the steroid application). Sera from individual mice werecollected on days 0, 3, 5, 7, 10 and 14 following primary immunization.The titer of anti-ovalbumin antibody was assessed by ELISA usingovalbumin for capture and HRPO-coupled, goat anti-murine Ig detectingantibodies with specificity for IgM and IgG subclasses. Each ELISA assaywas controlled with sera known to be positive or negative foranti-ovalbumin activity.

The results, shown in FIG. 14, establish that a topical application ofDHEA prior to immunization through the same skin site, provided the agedanimals with the ability to generate completely normal humoral immuneresponses. The untreated group of aged animals, and aged animalsprovided with topical DHEA on the footpads opposite the site ofimmunization, responded quite poorly to immunization, with minimalantibody being observed.

The results of reversing the age-related decline in humoral responseshas been repeated with BALB/c mice, and with C3H/HeN strain of mice.

These results establish that the pronounced lymphoid organ-specificchanges in the types of lymphokines produced by T cells from agedanimals given topical DHEA, can be paralleled by an equally dramaticenhancement in ability to generate potent humoral immune responses tochallenge with a foreign antigen protein.

Example 12 Lymphokine Production and Contact Hypersensitivity Responsesare Modulated in Thermally-Injured Mice

Some of the most profound immunological changes that appear as a resultof thermal injury are both rapid loss in the ability to develop cellularimmune responses of several types and an inability of activated Tlymphocytes to produce IL-2 and γ-IFN. In order to establish the effecton IL-4, the production of this lymphokine by T lymphocytes fromthermally-injured and control mice was examined.

Six to eight 8 week old BALB/c mice were shaved on their dorsal surfacesurfaces as a preparation for receiving a thermal injury. Two days afterremoval of truncal fur, all of the experimental mice were anaesthetized,and half were given a 20% total body surface area (TBSA), full-thicknessscald burn. Following revival from anesthesia, the burned mice werefluid resuscitated over a 3-day period using normal saline. Boththermally-injured and control groups of mice were allowed to feed anddrink normally for 5 days, at which time all animals were sacrificed.Splenocytes from individual thermally-injured and control mice wereprepared for culture in serum-free media. 1'10⁷ cells was dispensed, intriplicate, into 24-well culture plates with or without 1.5 μgmonoclonal anti-CD3ε. Culture plates were incubated in a 38° C., 10%CO₂, humidified chamber for 24 hours prior to collection of cell-freesupernatants for quantitative evaluation of IL-2, γ-IFN, and IL-4.Assays for each of these lymphokines was as described above.

The effect of the thermal injury on lymphokine production is shown inFIG. 15A. The results on the depressed production of IL-2 and γ-IFNagrees with other reports on humans and rodents. In contrast to thisobserved depression in IL-2 and γ-IFN levels, activated T cells fromthermally-injured mice were found to produce a greater amount of IL-4,as compared to activated splenocytes isolated from control mice.

Because thermal injury is known to compromise development of contacthypersensitivity responses, groups of thermally-injured (20% TBSA) andcontrol mice were contact sensitized to DNFB to demonstrate that thethermal injury protocol, in addition to reducing the capacity ofactivated T cells to produce IL-2 and γ-IFN, also results in a reducedability of injured mice to develop cellular immune responses. Five daysafter receiving a 20% TBSA, equivalent groups of normal andthermally-injured mice were sensitized by the application of DNFB totheir shaved abdomens. All experimental animals were challenged 4 dayslater by topical applications of DNFB to the right hind footpad. Theintensity of the hypersensitivity reaction was determined byquantitating the difference between thicknesses of the challenged andthe unchallenged footpad.

FIG. 15B shows the results of the DNFB challenge studies (the barsrepresent the mean±SEM). The results confirm that the development ofcontact sensitivity responses and production of the lymphokines whichare associated with promoting these responses are depressed in micegiven a 20% TBSA, full-thickness scald burn. Furthermore, the ability ofT cells to produce the lymphokine, IL-4, which promotes B-celldifferentiation, immunoglobulin isotype-switching, and also potentanti-inflammatory activity, is not apparently depressed as a result ofthermal injury. These findings suggest that thermal injury is aselective modulator of T-cell function.

Example 13 Treatment in vitro of Splenocytes from Thermally-Injured Micewith DHEA Restores the Capacity to Produce Lymphokines

The following study was performed in order to evaluate whether theexposure of T cells from thermally-injured mice to DHEA would influencetheir capacity to produce lymphokines subsequent to activation. Groupsof 4 to 6 BALB/c mice were either thermally injured with a 20% TBSAscald burn or were non-injured controls. Five days after thermal injury,the time when the "immunosuppression" is maximal, all surviving micewere sacrificed and splenocytes from individual mice were prepared forculture in serum-free media. Splenocytes were seeded into 24-wellmacroculture plates at 1×10⁷ cells/ml/well. Parallel cultures from eachmouse were sham or DHEA pulsed at 10⁻⁷ M for 60 minutes, washed multipletimes to remove nonbound steroid, and then were incubated in serum-freemedium and were either unstimulated or stimulated with 1.5 μg anti-CD3ε.Following a 24-hour incubation period, cell-free supernatants werequantitatively analyzed for IL-2 using the standard HT-2 bioassay, andfor γ-IFN, IL-4, IL-5, IL-3, and GM-CSF by capture Elisa. The resultsare shown in FIGS. 16A-16F, where the bars represent the ± standarddeviation for each lymphokine.

As seen from FIGS. 16A-16F, a comparison between lymphokines produced byactivated splenocytes from thermally-injured and control animalsindicates that thermal injury causes a depression in the capacity ofactivated T cells to secrete IL-2, γ-IFN, IL-3, and GM-CSF. Only minimalchanges (elevations) in the quantities of IL-4 and IL-5 were observed.The treatment of T cells from the thermally-injured animals with DHEAreversed the inhibitory effect on IL-2, γ-IFN, IL-3, and GM-CSFproduction; the values of these lymphokines returned to near controllevels. The DHEA treatment had no effect on IL-4 or IL-5 production.

Example 14 Treatment in vivo of Thermally Injured Mice with DHEAPreserves Normal Immune Function

The following study illustrates that the direct administration of DHEAto mice shortly after thermal injury influences their levels ofimmunocompetence.

Groups of 12 thermally-injured and 6 control BALB/c mice wereestablished as described above. After subjecting the mice to a 20% TBSAscald burn, six of the thermally-injured mice were given a subcutaneousinjection of 100 μg DHEA in a propylene glycol carrier. All remaininganimals received the carrier alone. Five days later, all surviving micewere sacrificed and their splenocytes were individually prepared forculture, and activated with anti-CD3ε to induce lymphokine secretion.Culture supernatants were collected 24 hours after activation andevaluated for lymphokine content, as described above. The results of thestudy are presented in FIGS. 17A-17F, where the bars represent mean ±SDfor each value. As seen in FIGS. 17A-17F, DHEA directly influences IL-2,γ-IFN, IL-3, and GM-CSF production by T cells isolated fromthermally-injured mice. The administration of a single bolus injectionof DHEA (100 μg) 1 hour after thermal injury was sufficient to preservefor at least 5 days a normal capacity by their lymphocytes to produceIL-2, γ-IFN, IL-3, and GM-CSF following activation. No significantchanges from normal were observed in the levels of IL-4 and IL-5 made byactivated lymphoid cells from these animals.

The effect of DHEA treatment in vivo on the animals' development ofcellular immune responses was examined. Parallel groups ofthermally-injured and control mice were either given 100 μg DHEA inpropylene glycol carrier or the carrier alone 1 hour post burn. Theseanimals were contact sensitized 5 days later by administration of DNFBon the abdomen. Challenge doses of DNFB to the right footpads wereapplied 4 days later. The differences in thickness between the right(challenged) and the left (unchallenged) footpads were used toquantitate the contact hypersensitivity responses. The results are shownin FIG. 18; the bars represent mean ±SD for each group of mice. As shownin FIG. 18, the intensity of the contact hypersensitivity responseselicited by thermally-injured mice are markedly depressed as compared tocontrols. The administration of DHEA to thermally-injured mice was foundto completely preserve the ability of these animals to develop contacthypersensitivity responses of normal intensity.

We conclude from these studies that DHEA treatment post burn is aneffective therapy for preserving the capacity of T-lymphocytes fromthermally-injured animals to produce normal quantities of a number oflymphokines, especially those that are essential for development ofcellular immune responses. This finding is supported by the additionaldemonstration that DHEA-treated thermally-injured mice also retain theircapacity to develop normal contact hypersensitivity responses.

Example 15 DHEA Treatment in vivo Promotes Resistance to Infection by L.monocytogenes in Thermally Injured Mice

This study addresses the utility of DHEA therapy post burn in preservingresistance to a bacterial infection. C3H/HeN strain mice are inherentlyresistant to infection by the gram positive intracellular pathogen, L.monocytogenes. However, thermal injury results in an increasedsusceptibility to this pathogen. Therefore, a switch from "resistant" toa more "susceptible" phenotype provides a model system to evaluate theeffect of DHEA on preserving the "resistant" phenotype inthermally-injured animals.

Normal (control) and thermally-injured mice were prepared as describedabove. Half of the thermally-injured mice received a single bolusinjection of 100 μg DHEA subcutaneously within 1 hour after thermalinjury. Three days later, all mice were infected with 2×10⁶ viable L.monocytogenes organisms, and 3 days after infection the mice weresacrificed and homogenates of individual spleens were prepared. Thenumber of colonies of L. monocytogenes per spleen were evaluated usingstandard methodology, and scored. The results are presented graphicallyin FIG. 19, where the bars represent means ±SEM for each treatmentgroup. The results indicate that thermal injury enhances thesusceptibility of the C3H strain mice to infection by L. monocytogenes.Of consequence, DHEA treatment of burned animals not only preserves theresistant phenotype, but surprisingly, the level of resistance toinfection is actually enhanced by DHEA treatment over that observed inthe control group.

Example 16 The Effect of 16α-bromo-DHEA and 16α-chloro-DHEA onAge-Associated Changes in T-cell Lymphokine Production

In order to demonstrate the effectiveness of the cogeners of DHEA,16α-bromo-DHEA and 16α-chloro-DHEA on age-associated changes in T-celllymphokine production, the following study was performed. Suspensionscontaining T cells were prepared from the mesenteric lymph node and fromthe spleen of groups of aged (old) mice. The T cell suspensions wereexposed in vitro to DHEA, 16α-bromo-DHEA or 16α-chloro-DHEA at aconcentration of 10⁻⁷ M for 60 minutes. After activation with anti-CD3ε,the IL-2 titers in the cell-free supernatants was measured by bioassay.The results, shown in FIG. 20, indicate that 16α-bromo-DHEA is as activeas DHEA in restoring IL-2 production by the activated T-cells. However,surprisingly, 16α-chloro-DHEA had little, if any, effect on restoringIL-2 production.

Example 17 The Effect of 16α-bromo-DHEA and 16α-chloro-DHEA on theDepressed Humoral Immune Responses of Aged Mice to Protein Antigens

The effectiveness of the cogeners of DHEA, 16α-bromo-DHEA and16α-chloro-DHEA on age-associated changes in the humoral immune responseis demonstrated in the following study. Groups of mature (young) andaged (old) mice were treated by topical administration of 10 μg of DHEA,16α-bromo-DHEA, or 16α-chloro-DHEA. Three hours subsequent to treatment,the treated and control animals were subjected to an ovalbumin (OVA)challenge as described in the Examples above, and the anti-ovalbuminantibody response was measured at 0, 3, 5, 7, 10, and 14 days afterimmunization. The results, shown in FIG. 21, indicate that16α-bromo-DHEA (BrD) is as effective as DHEA in restoring humoral immuneresponsiveness. The chlorinated cogener, 16α-chloro-DHEA (ClD) yielded alower, but significant effect on antibody production.

We claim:
 1. A method for treating naturally occurring age-relateddecline in immune function in a human, said decline resulting from (i)an age-related increase in production of IL-4 or IL-5 or (ii) anage-related decrease in production of IL-2, IL-3, γ-IFN, GM-CSF orantibody, wherein said method comprises administering to said human anantigen and DHEA-S in an amount effective to (i) decrease production ofIL-4 or γ-IFN or (ii) increase production of IL-2, IL-3, IL-5, GM-CSF orantibody.
 2. The method of claim 1 wherein said compound is administeredparenterally.
 3. The method of claim 1 wherein said compound isadministered transdermally.
 4. The method of claim 1 wherein saidcompound is administered transmucosally.
 5. A method for normalizing aspecific immune response in a human with an acquired or transient stateof immune compromise, immune deficiency or immune dysregulation whereinsaid immune compromise, deficiency or dysregulation results frominfection, stress, burns, surgery or autoimmune disease, saidnormalizing of said specific immune response being (i) an increasedproduction of one or more of IL-2, IL-3, γ-IFN, GM-CSF or antibody or(ii) a decreased production of one or more of IL-4 or IL-5, wherein saidmethod comprises administering to said human an antigen and DHEA-S in anamount effective to (i) increase production of IL-2, IL-3, γ-IFN, GM-CSFor antibody or (ii) decrease production of IL-4 or IL-5.
 6. The methodof claim 5 wherein said human is immunodeficient due to stress.
 7. Themethod of claim 5 wherein said human is immunodeficient due to trauma.8. The method of claim 5 wherein said compound and said antigen are eachadministered such that the compound and antigen will drain to the samelymph node.
 9. A method for treating naturally occurring age-relateddecline in immune function in a human, said decline resulting fromage-related changes causing (i) an increased production of IL-4 or IL-5or (ii) a decreased production of IL-2, IL-3, γ-IFN, GM-CSF or antibody,wherein said method comprises administering to said human DHEA-S in anamount effective to (i) decrease production of IL-4 or IL-5 or (ii)increase production of IL-2, IL-3, γ-IFN, GM-CSF or antibody.
 10. Themethod of claim 9 wherein said compound is administered parenterally.11. The method of claim 9 wherein said compound is administeredtransdermally.
 12. The method of claim 9 wherein said compound isadministered transmucosally.
 13. A method for normalizing a specificimmune response in a human with an acquired or transient state of immunecompromise, immune deficiency or immune dysregulation wherein saidimmune compromise, deficiency or dysregulation results from infection,stress, burns, surgery or autoimmune disease, said normalizing of saidspecific immune response being (i) an increased production of IL-2,IL-3, γ-IFN, GM-CSF or antibody or (ii) a decreased production of IL-4or IL-5, wherein said method comprises administering to said humanDHEA-S in an amount effective to (i) increase production of IL-2, IL-3,γ-IFN, GM-CSF or antibody or (ii) decrease production of IL-4 or IL-5.14. The method of claim 13 wherein said human is immunodeficient due tostress.
 15. The method of claim 13 wherein said human is immunodeficientdue to trauma.
 16. A method for restoring contact hypersensitivityresponse in a human with an acquired or transient state of immunecompromise, immune deficiency or immune dysregulation wherein saidimmune compromise, deficiency or dysregulation results from infection,stress, burns, surgery or autoimmune disease, wherein said methodcomprises administering to said human DHEA-S in an amount effective torestore contact hypersensitivity.
 17. A method for restoring contacthypersensitivity response in a human with an acquired or transient stateof immune compromise, immune deficiency or immune dysregulation whereinsaid immune compromise, deficiency or dysregulation results frominfection, stress, burns, surgery or autoimmune disease wherein saidmethod comprises administering to said human an antigen and DHEA-S in anamount effective to restore contact hypersensitivity.
 18. A method fortreating naturally occurring age-related decline in immune function in amammal, said decline resulting from (i) an age-related increase inproduction of IL-4 or IL-5 or (ii) an age-related decrease in productionof IL-2, IL-3, γ-IFN, GM-CSF or antibody, wherein said method comprisesadministering to said mammal an antigen and DHEA-S in an amounteffective to (i) decrease production of IL-4 or IL-5 or (ii) increaseproduction of IL-2, IL-3, γ-IFN, GM-CSF or antibody.
 19. The method ofclaim 18 wherein said compound is administered parenterally.
 20. Themethod of claim 18 wherein said compound is administered transdermally.21. The method of claim 18 wherein said compound is administeredtransmucosally.
 22. A method for normalizing a specific immune responsein a mammal with an acquired or transient state of immune compromise,immune deficiency or immune dysregulation wherein said immunecompromise, deficiency or dysregulation results from infection, stress,burns, surgery or autoimmune disease, said normalizing of said specificimmune response being (i) an increased production of one or more ofIL-2, IL-3, γ-IFN, GM-CSF or antibody or (ii) a decreased production ofone or more of IL-4 or IL-5, wherein said method comprises administeringto said mammal an antigen and DHEA-S in an amount effective to (i)increase production of IL-2, IL-3, γ-IFN, GM-CSF or antibody or (ii)decrease production of IL-4 or IL-5.
 23. The method of claim 22 whereinsaid mammal is immunodeficient due to stress.
 24. The method of claim 22wherein said mammal is immunodeficient due to trauma.
 25. The method ofclaim 22 wherein said compound and said antigen are each administeredsuch that the compound and antigen will drain to the same lymph node.26. A method for treating naturally occurring age-related decline inimmune function in a mammal, said decline resulting from age-relatedchanges causing (i) an increased production of IL-4 or IL-5 or (ii) adecreased production of IL-2, IL-3, γ-IFN, GM-CSF or antibody, whereinsaid method comprises administering to said mammal DHEA-S in an amounteffective to (i) decrease production of IL-4 or IL-5 or (ii) increaseproduction of IL-2, IL-3, γ-IFN, GM-CSF or antibody.
 27. The method ofclaim 26 wherein said compound is administered parenterally.
 28. Themethod of claim 26 wherein said compound is administered transdermally.29. The method of claim 26 wherein said compound is administeredtransmucosally.
 30. A method for normalizing a specific immune responsein a mammal with an acquired or transient state of immune compromise,immune deficiency or immune dysregulation wherein said immunecompromise, deficiency or dysregulation results from infection, stress,burns, surgery or autoimmune disease, said normalizing of said specificimmune response being (i) an increased production of IL-2, IL-3, γ-IFN,GM-CSF or antibody or (ii) a decreased production of IL-4 or IL-5,wherein said method comprises administering to said mammal DHEA-S in anamount effective to (i) increase production of IL-2, IL-3, γ-IFN, GM-CSFor antibody or (ii) decrease production of IL-4 or IL-5.
 31. The methodof claim 30 wherein said mammal is immunodeficient due to stress. 32.The method of claim 30 wherein said mammal is immunodeficient due totrauma.
 33. A method for restoring contact hypersensitivity response ina mammal with an acquired or transient state of immune compromise,immune deficiency or immune dysregulation wherein said immunecompromise, deficiency or dysregulation results from infection, stress,burns, surgery or autoimmune disease, wherein said method comprisesadministering to said mammal DHEA-S in an amount effective to restorecontact hypersensitivity.
 34. A method for restoring contacthypersensitivity response in a mammal with an acquired or transientstate of immune compromise, immune deficiency or immune dysregulationwherein said immune compromise, deficiency or dysregulation results frominfection, stress, burns, surgery or autoimmune disease wherein saidmethod comprises administering to said mammal an antigen and DHEA-S inan amount effective to restore contact hypersensitivity.